Membrane depolarization resulting from inhibition of background K+ channels is a widespread but poorly understood modulatory mechanism that increases activity of excitable cells. Metabotropic receptors that are capable of mediating such a slow depolarization have been identified for most major transmitters. This represents perhaps the predominant mechanism for slow synaptic excitation in the brain and may contribute to positive chronotropic effects in the heart. The molecular identity of the K+ channel(s) targeted for inhibition is unknown in most systems, but in a number of cases Kir3.x (GIRK) family channels are implicated. This is particularly interesting since activation of the same GIRK channels by receptors utilizing PTx-sensitive pathways can mediate a slow membrane hyperpolarization. This dual modulation of GIRKs provides a mechanism for dynamic control of membrane potential in the same cell by different classes of receptor targeting the same ion channel. The cellular and molecular mechanisms of GIRK activation are well-described. By contrast, there is little information regarding mechanisms that underlie receptor-mediated inhibition of GIRK channels. We have developed a model system, based on heterologous expression in mammalian cells of the thyrotropin-releasing hormone (TRH) receptor with GIRK-1 and -4, that recapitulates features of the dual modulatory mechanism. We use this system and the tools of molecular biology to manipulate individual components of the pathway in order to test the hypothesis that specific G protein subunits, distinct from those involved in GIRK activation, are critical to support receptor-mediated GIRK inhibition. The specific aims are: [1] Determine which G protein subunits are capable of causing inhibition of GIRKs. Specific Galpha subunits or Gbetagamma dimers are tested to determine if they: a) inhibit GIRK currents in whole cell assays; b) bind directly to purified GIRK protein domains; and c) inhibit GIRK channels in excised patches. [2] Determine which domains of Gbeta are important for GIRK inhibition and activation. Chimeric and site-directed mutants of Gbeta1 and Gbeta5 are used to identify regions that support GIRK channel modulation. [3] Determine G protein subunits that mediate GIRK inhibition by TRH receptors. Signaling by Galpha and/or Gbetagamma subunits is disrupted to determine which subunit supports receptor-mediated GIRK inhibition. [4] Determine second messenger pathway(s) that mediate GIRK inhibition by TRH receptors. Pharmacological tools are used to perturb second messenger pathways hypothesized to mediate GIRK channel inhibition by TRH receptors. These experiments will provide important information regarding not only mechanisms of K+ channel modulation that underlie slow synaptic excitation, but also regarding determinants of G protein-effector signaling specificity.